Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors-including toxic byproducts from biomass pretreatment and poor fermentation of xylose and other pentose sugars-currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocellulosic hydrolysate or tolerance to stress conditions relevant to industrial biofuel production, through genome and transcriptome sequencing analysis. All stress resistant strains were highly mosaic, suggesting that genetic admixture may contribute to novel allele combinations underlying these phenotypes. Strain-specific gene sets not found in the lab strain were functionally linked to the tolerances of particular strains. Furthermore, genes with signatures of evolutionary selection were enriched for functional categories important for stress resistance and included stress-responsive signaling factors. Comparison of the strains’ transcriptomic responses to heat and ethanol treatment-two stresses relevant to industrial bioethanol production-pointed to physiological processes that were related to particular stress resistance profiles. Many of the genotype-by-environment expression responses occurred at targets of transcription factors with signatures of positive selection, suggesting that these strains have undergone positive selection for stress tolerance. Our results generate new insights into potential mechanisms of tolerance to stresses relevant to biofuel production, including ethanol and heat, present a backdrop for further engineering, and provide glimpses into the natural variation of stress tolerance in wild yeast strains.